1. Field
This disclosure is concerned generally with a microbial culture device and a method of determining whether a sample of an unknown microbe can be deemed gram positive or gram negative.
2. Prior Art
All bacteria may be classified as either gram positive or gram negative depending on the staining properties of the cells. The gram stain reaction may be correlated with a large number of other properties and it is perhaps the single most important piece of information which may be communicated about a given species of bacteria. See, for example, Lammanna, C. and Mallette, M., Basic Bacteriology, 3rd Ed., Williams (1965). Wilkins, Baltimore, Maryland, pp. 162-164, (1965 ).
Very generally, the determination of whether a given microbe is gram positive or gram negative is performed as follows: a sample of an unknown colony is smeared on a microscope slide, a drop of water is added, and the sample is permitted to dry on the slide. The brief use of a flame causes the bacteria to stick to the slide. Then a crystal violet solution is added to the specimen, after which the specimen appears purple. This is followed by the addition of an iodine solution. After this step, the entire sample still appears purple. At this point, however, the stain of gram positive samples is immobilized within cell walls whereas the stain of gram negative remains free within the cell walls. The slide is then washed with alcohol until no more purple stain comes off with the washings. At this point, a gram positive sample, having stain immobilized in the cell walls, remains purple. A gram negative sample becomes colorless. The slide is then counterstained with safranin (red dye) after which a gram positive sample remains purple while a gram negative sample becomes red. The test is completed by washing the slide with water, drying it, and observing the specimen with a microscope to determine the specimen color.
Although the above steps themselves may appear to be relatively simple, it is known that the proper execution and interpretation of the gram stain requires considerable degrees of training and skill. Further, the test requires the availability of a high-power microscope and takes at least 5 minutes beyond sample culture time. In addition, the gram staining properties of some bacteria are dependent on the age of the culture. Also, the capacity of bacteria to grow on various selective media often correlates with the gram staining property. For example, incorporation into culture media of certain dyes, such as crystal violet or brilliant green, tends to inhibit the growth of gram positive bacteria more than that of the gram negative microbes. On the other hand, the incorporation of phenylethyl alcohol into media tends to inhibit gram negative organisms more than gram positive species. In addition to the known disadvantages associated with present methods for determining gram properties, it is known that the effects of growth inhibitors is only relative (i.e., the favored organisms are inhibited somewhat even under permissive conditions and several different inhibitory media are required to examine an organism of unknown gram properties.)
Although the exact chemical and/or physical mechanism of the gram staining procedure is not completely understood, it is known that the cell walls of gram positive and gram negative organism have significant chemical differences. For example, cell walls of gram positive microbes comprise about 90% peptidoglycan with the remaining constituents comprising teichoic acid, lipids, polysaccharides, and proteins. On the other hand, cell walls of gram negative microbes comprise about 5 to 20% peptidoglycan with the remaining constituents comprising lipids, polysaccharides, and proteins. See, for example, Brock, T. D. Biology of Microorganisms, 2nd Ed., Prentice-Hall (1974). In general, gram negative organisms have significantly more lipids in their cell walls than gram positive species.
It is known that certain lipophilic fluorescent probes such as 8-anilino-1-naphthalene sulfonate can be used to study various cell membranes. See, for example, T. A. Pederson and A. Lode, Arch. Mikrobiol. 77, 118-126 (1971); ); W. A. Cramer and S. K. Phillips, J. Bacteriol., 104(2), 819-825 (1970); and R. B. Freedman et al., Probes Struct. Funct. Macromol. Membranes, Proc. Colloq. Johnson Res. Found., 5th 1969 (Pub. 1971), 1, 325-338 (Eng.). We are unaware, however, of any disclosure which makes use of such probes to determine whether a given microbe has gram positive or gram negative properties.
Quite surprisingly, we have found that differences in cell wall lipid amounts can be used to distinguish gram positive and gram negative organisms. Our method avoids many of the problems associated with present gram stain methods. Details of our method and devices for using it are disclosed herein.